Características y relevancia en patogénesis de la actividad canal iónico de la proteína E del coronavirus causante del síndrome respiratorio agudo grave
Author
Nieto Torres, José LuisEntity
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Date
2014-06-23Subjects
Aparato respiratorio - Enefermedades - Tesis doctorales; Infecciones por coronavirus - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-06-2014Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Several
highly
pathogenic
viruses
encode
at
least
one
protein
displaying
ion
channel
(IC)
activity.
These
proteins,
named
viroporins,
play
central
roles
in
virus
life
cycle
and
may
promote
pathogenesis.
Severe
acute
respiratory
syndrome
coronavirus
(SARS-‐CoV)
was
the
etiological
agent
of
a
worldwide
epidemic
affecting
8000
people
and
causing
10%
death
rate.
Previously,
our
laboratory
generated
a
SARS-‐CoV
mutant
lacking
the
multifunctional
envelope
(E)
protein,
which
exhibits
viroporin
properties,
as
an
attenuated
vaccine
candidate.
In
this
Thesis,
we
have
combined
multidisciplinary
approaches
to
characterize
E
protein,
its
IC
activity
and
the
specific
contribution
of
this
function
to
SARS-‐CoV
virulence.
Upon
generation
of
specific
monoclonal
and
polyclonal
antibodies,
E
protein
was
localized
at
the
ERGIC
of
infected
cells,
showing
the
structural
properties
(topology
and
oligomerization)
required
for
ion
channel
activity.
Electrochemical
measurements
revealed
that
SARS-‐CoV
E
protein
behaved
as
a
non-‐voltage
gated
protein-‐lipid
pore,
whose
selectivity
and
conductance
were
largely
controlled
by
the
charge
of
the
lipid
membranes.
In
addition,
mutations
that
inhibited
ion
channel
activity
were
identified.
Recombinant
mouse
adapted
SARS-‐CoVs
containing
these
mutations
were
generated
by
reverse
genetics.
Although
the
growth
of
the
resultant
viruses
was
not
significantly
compromised,
these
viruses
tended
to
evolve
restoring
IC
activity.
Molecular
modeling
revealed
that
the
amino
acid
substitutions
incorporated
by
the
mutant
viruses
may
compensate
the
structural
deficiencies
caused
by
the
mutation
suppressing
ion
conductivity,
rendering
active
channels.
Interestingly,
viruses
lacking
E
protein
IC
activity
were
attenuated
in
the
mouse
model.
Suppression
of
E
protein
IC
activity
led
to
a
significant
reduction
of
lung
pathology
caused
by
SARS-‐CoV,
reflected
in
a
diminished
lung
edema,
the
ultimate
cause
of
death.
Lack
of
edema
correlated
with
proper
localization
of
Na+/K+
ATPase
within
the
epithelia,
which
facilitates
edema
resolution.
In
addition,
a
reduction
of
the
inflammatory
response
driven
by
key
cytokines
involved
in
disease
progression,
such
as
inflammasome
activated
IL-‐1β,
TNF
and
IL-‐6
was
associated
to
the
absence
of
E
protein
IC
activity.
This
work
provides
key
information
on
the
relevance
of
ion
channel
activity
from
viral
proteins
in
virulence,
an
observation
that
could
be
extended
to
other
highly
pathogenic
coronaviruses
such
as
MERS-‐CoV
or
to
other
viruses.
Interestingly,
virus
IC
activity
could
be
a
target
for
therapeutic
interventions.
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